The ability to control hybridization of a nucleic acid strand (a probe) to its complement, while excluding imperfectly base-paired probe hybridization has been central to the advancement of both molecular biological techniques and to design of nucleic acid diagnostic systems. Much attention has been paid to this issue because identification of a particular mutant nucleic acid sequence fully complementary to a probe can permit detection of, for example, the existence of a mutant sequence (genetic disorders) or a particular virulent bacterial or viral strain in a patient. Thus, is studies of the physics of mismatched probe:target energetics has focused on the difference in free energy of such mismatches with the hope that such knowledge will benefit the development of assays in which such mismatches are excluded. For example, in diagnosis of a genetic disorder a mutant probe for targeting a nucleic acid molecule having a sequence containing a single base mismatch associated with such a disorder would produce a false positive result if the probe also hybridizes to the wild-type (unmutated) sequence. In other words, the assay must be sufficiently discriminatory in order for the probe to bind to the molecule having the mutated base and not to the molecule lacking the mutated base. If the probe hybridizes to both molecules, then the hybridization result would indicate the presence of the single base mutated sequence even though it was not, in fact, contained in the tested sample. In general, the environment of the reaction is manipulated to eliminate such mismatch probe:target interactions by modifying the physical conditions for hybridization (e.g., temperature and or time) or composition of the hybridization buffer (e.g., salt, divalent ions denaturing agents, etc.).
On the other hand, however, it is advantageous, in some applications, to have a probe which is known to hybridize with molecules containing particular mismatched sequences (a “marginally indiscrimninant” probe) within a desired degree of homology to the probes' perfect complement. This would permit a single probe to be used in an assay for determining the presence of nucleic acid molecules containing any of the mismatched sequences. Such an assay would thus reduce, or possibly even eliminate, the need for more than one probe, each containing a nucleic sequence precisely corresponding to a sequence of a target molecule. Achievement of such a probe could be useful as a “multiplex” (multiple assays from one probe) probe. To date, for example, conventional multiplexing has relied upon the inclusion of multiplex specific probes into one cocktail reaction (e.g., multiplex polymerase chain reaction (PCR)), rather than just one probe.
There are always going to be constraints on an indiscrimninant probe. It would be generally acceptable for a probe to hybridize to any nucleic acid molecule whether complementary or not (although there may be limited use for such a probe in detecting the presence or absence of any DNA). This type of probe and/or conditions for hybridization of the probe would detect even sequences which shared no homology with the probes' complement. At the other end of the spectrum, it is desirable that when designing a marginally indiscrimninant probe for detecting viral nucleic acid sequences, for example, to design a probe such that a single probe will pick up all known of sequence within a limited degree of homology (say 10, 20, 30, 40 or 50% homology).
There are known approaches for detecting target nucleic acids by hybridization of a probe having a nucleic acid sequence fully complementary to or substantially complementary to a sequence of a target nucleic add. Methods have thus been developed to detect viral nucleic acid sequences and their variants by hybridization using probes fully complementary to or substantially complementary to the viral nucleic acid sequences, as exemplified by U.S. Pat. Nos. 5,008,182; 5,079,351; 5,268,268; 5,567,603; 5,594,122; 5,594,123; 5,599,662; and 5,733,781, the text of which is incorporated herein by reference.
The specifications of these patents disclose methods and compositions of nucleic acids for as probes for detecting nucleic acid sequences of the family of Human T-cell Leukemia Viruses (HTLV) and the Human Immunodeficiency Virus (HIV). HIV and its variants are thought to be responsible for the acquired immunodeficiency syndrome (AIDS). The probes and methods disclosed in these patents for detecting the presence or absence of the viral DNA utilize probes to conserved regions of these viruses, but the disclosed approaches have limited applicability. This is because of the now well-known genetic variability of human immunodeficiency viruses. Genetic variations arise with high frequency. This variability has complicated the development of assays for detecting the presence of their genetic material. Further, while a comparison of various HIV-1 isolates has revealed, regions of the genome that are reasonably well conserved, it is possible that even the conserved regions, regions to which the probes have been designed to hybridize, may at mutate in the future. If so, probes designed for detecting the conserved regions may not hybridize to the one is conserved region as a result of base mismatches.
As a further example, U.S. Pat. No. 5,567,603 describes probes for detecting HIV-3 that hybridize neither with the sequences of HIV-1 nor with the sequences of HIV-2 under stringent hybridization conditions. Thus, the ability to design a single nucleic acid probe and a method that will allow hybridization of the probe to all HIV strains and their variants but not to other non-target partially complementary nucleic acid sequences or other non-related viral nucleic acid sequences would have advantages over current approaches.